Genetic screen identifies non-mitochondrial proteins involved in the maintenance of mitochondrial homeostasis

The mitochondrial unfolded protein response (UPR mt ) is an important stress response that ensures the maintenance of mitochondrial homeostasis in response to various types of cellular stress. We previously described a genetic screen for Caenorhabditis elegans genes, which when inactivated cause UPR mt activation, and reported genes identified that encode mitochondrial proteins. We now report additional genes identified in the screen. Importantly, these include genes that encode non-mitochondrial proteins involved in processes such as the control of gene expression, post-translational modifications, cell signaling and cellular trafficking. Interestingly, we identified several genes that have been proposed to participate in the transfer of lipids between peroxisomes, ER and mitochondria, suggesting that lipid transfer between these organelles is essential for mitochondrial homeostasis. In conclusion, this study shows that the maintenance of mitochondrial homeostasis is not only dependent on mitochondrial processes but also relies on non-mitochondrial processes and pathways. Our results reinforce the notion that mitochondrial function and cellular function are intimately connected.

. Genetic screen identifies additional proteins involved in the maintenance of mitochondrial homeostasis 5/11/2022 -Open Access A. The candidates identified in the screen are listed by functional groups. When available, the name of the human ortholog as stated on Alliancegenome.org is indicated. Protein localization is indicated and is based on experimental evidence in C. elegans or experimental evidence using the human ortholog. In case no experimental evidence was available, the localization was predicted with the PSORT II website (https://psort.hgc.jp/form2.html). Newly identified candidates are indicated in green whereas candidates identified in previous genetic screens are indicated in blue (Runkel et al. 2013, Bennett et al. 2014. Finally, candidates identified in a previous genetic screen for enhancers of fzo-1(tm1133lf)-induced UPR mt are indicated in yellow (Haeussler et al. 2021). B. DIC and fluorescence images of animals carrying the hsp-6p::GFP (zcIs13) reporter and either homozygous for the C25H3.11 loss-of-function allele ok2632 (C25H3.11(ok2632)) or homozygous wildtype for C25H3.11 (mIn1/mIn1). The balancer mIn1 carries the dpy-10(e128) mutation and the pharyngeal myo-2p::GFP transgene, which is visible in the mIn1/mIn1 Dumpy animal (indicated by an arrow in the right panel). Induction of the hsp-6p::GFP reporter is visible in the intestine of C25H3.11(ok2632) homozygous animals (indicated by an arrowhead in the right panel) but not in animals homozygous for the balancer mIn1. C. Wild-type (+/+) and C25H3.11(ok2632) animals were analyzed by Western analysis using anti-tubulin (loading control) and anti-GFP antibodies. D. C25H3.11 VPS13D has been proposed to be a conduit that transfers lipids between ER and mitochondria and between ER and peroxisomes (Guillen-Samander et al. 2021). It localizes to the ER in a VPR-1 VAP-B dependent manner (Guillen-Samander et al. 2021). PRX-3 PEX3 and PRX-12 PEX12 are essential for peroxisome biogenesis and hence peroxisomal beta oxidation (Petriv et al. 2002). Inactivation of any of these genes induces the expression of the hsp-6p::GFP reporter and hence triggers UPR mt , indicating that these proteins are required to maintain mitochondrial homeostasis.

Description
Mitochondrial UPR (UPR mt ) is a conserved unfolded protein stress response that is necessary for the maintenance of mitochondrial homeostasis in response to various types of stress (Shpilka et al. 2018). To systematically identify processes that trigger UPR mt when compromised, we performed a genome-wide RNAi screen in Caenorhabditis elegans (Rolland et al. 2019). To that end, we used a reporter construct, hsp-6p::GFP, the expression of which is induced in the intestine upon UPR mt activation (Yoneda et al. 2004). Using this approach, we identified 172 genes that when knocked-down induce the expression of the hsp-6p::GFP reporter (and hence activate UPR mt ) and encode mitochondrial proteins (Rolland et al. 2019). We now report an additional seven genes that fall into this category, which increases the number of genes identified encoding mitochondrial proteins to 179 ( Figure 1A). Furthermore, our screen also led to the identification of 19 genes that induce the expression of the hsp-6p::GFP reporter when knocked-down but encode non-mitochondrial proteins ( Figure 1A). In summary, our genetic screen identified in total 179 genes encoding mitochondrial proteins and 19 genes encoding nonmitochondrial proteins, all of which are required for the maintenance of mitochondrial homeostasis. The non-mitochondrial proteins identified have been shown or are predicted to localize to several sub-cellular compartments, including the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus and peroxisomes. This indicates that several cellular pathways including non-mitochondrial pathways participate in the maintenance of mitochondrial homeostasis.
Among the non-mitochondrial proteins identified, we further investigated C25H3.11, the ortholog of mammalian VPS13D (Vacuolar Protein Sorting-associated protein 13D). To confirm this candidate, we analyzed the effect of the C25H3.11 loss-offunction allele ok2632 on the expression of the hsp-6p::GFP reporter (ok2632 is a 1740bp deletion that removes part of exon 3 and 4 and creates a frameshift; it therefore most likely represents a null allele). Since animals homozygous for ok2632 are sterile, we maintained it over the balancer mIn1. Microscopy analysis revealed that the progeny of the balanced strain that is homozygous for ok2632 (C25H3.11(ok2632)) exhibits intestinal hsp-6p::GFP expression in contrast to animals homozygous for the balancer (mIn1/mIn1) ( Figure 1B). We confirmed this result by Western analysis (Figure 1C). Therefore, we conclude that the loss of C25H3.11 VPS13D induces UPR mt .
VPS13D was shown to mediate contact sites between mitochondria, ER and peroxisomes (Guillen-Samander et al. 2021). Specifically, VPS13D has been proposed to provide a lipid conduit between peroxisomes and ER as well as between ER and mitochondria. Interestingly, localization of VPS13D to the ER requires the protein VAP-B, the ortholog of another candidate identified in our screen, VPR-1(Guillen-Samander et al. 2021). Inactivation of these genes could affect mitochondrial metabolism by disrupting lipid transport between these organelles. Consistent with this idea, we identified other candidates such as prx-3 PEX3 or prx-12 PEX12, which are essential for peroxisome biogenesis and likely cause defects in lipid homeostasis when inactivated. In summary, we propose that the transfer of lipids between mitochondria, ER and peroxisomes is required to maintain mitochondrial homeostasis ( Figure 1D).
We previously showed that by causing a reduction in mitochondrial membrane potential (which is sensed by the UPR mt transcription factor ATFS-1), the impairment of most but not all mitochondrial processes activates UPR mt (Rolland et al. 2019).
Our new results reveal that non-mitochondrial processes can also activate UPR mt when compromised. We propose that impairment of these processes might affect mitochondrial membrane potential indirectly through, for example, defects in lipid transfer into and out of mitochondria, thereby inducing UPR mt .